bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Filleton, Abdul, et al. 1 Smc5/6-antagonism by HBx is an evolutionary-conserved function of hepatitis B virus 2 infection in mammals 3 4 Fabien Filleton1*, Fabien Abdul2*, Laetitia Gerossier3, Alexia Paturel3, Janet Hall3, Michel 5 Strubin2, Lucie Etienne1§ 6 7 1 CIRI – International Center for Infectiology Research, Inserm, U1111, Université Claude 8 Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, F- 9 69007, Lyon, France ; 2 Department of Microbiology and Molecular Medicine, University 10 Medical Centre (C.M.U.) / University of Geneva, Rue Michel-Servet 1, 1211 Geneva 4, 11 Switzerland ; 3 CRCL- UMR Inserm 1052 - CNRS 5286, 151 cours Albert Thomas, 69300 12 Lyon, France. 13 14 * Contributed equally to this work 15 §: Correspondence: Lucie Etienne, CIRI, ENS-Lyon, 46 Allée d’Italie, 69364 Lyon, France. 16 E-mail: [email protected] 17 18 Keywords: Smc5/6 complex, evolution of SMC genes, Hepatitis B virus, HBx, restriction 19 factor, antagonist, positive selection, virus-host interaction, genetic conflict 20 1 bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Filleton, Abdul, et al. 21 Abstract 22 Infection with Hepatitis B virus (HBV) is a major cause of liver disease and cancer in 23 humans. HBVs (family Hepadnaviridae) have been associated with mammals for millions of 24 years. Recently, the Smc5/6 complex, known for its essential housekeeping functions in 25 genome maintenance, was identified as an antiviral restriction factor of human HBV. The 26 virus has however developed a counteraction mechanism by degrading the complex via its 27 regulatory HBx protein. Whether the antiviral activity of the Smc5/6 complex against 28 hepadnaviruses is an important and evolutionary-conserved function is unknown. Here, we 29 used a combined evolutionary and functional approach to address this question. We first 30 performed phylogenetic and positive selection analyses of the six Smc5/6 complex subunits 31 and found that they have been highly conserved in primates and mammals. Yet, the Smc6 32 subunit showed marks of adaptive evolution, potentially reminiscent of virus-host “arms- 33 race”. We then functionally tested the HBx from six very divergent hepadnaviruses now 34 naturally infecting primates, rodents, and bats. Despite little sequence homology, we 35 demonstrate that these HBx efficiently degraded mammalian Smc5/6 complexes, 36 independently of the host species and of the sites under positive selection. Importantly, all 37 also rescued the replication of an HBx-deficient HBV in primary human hepatocytes. These 38 findings point to an evolutionary-conserved requirement for Smc5/6 inactivation by HBx, 39 showing that the Smc5/6 antiviral activity has been an important defense mechanism against 40 hepadnaviruses in mammals. Interestingly, Smc5/6 may further be a restriction factor of other 41 yet unidentified viruses that have driven some of its adaptation. 42 2 bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Filleton, Abdul, et al. 43 Importance 44 Infection with hepatitis B virus (HBV) led to 887000 human deaths in 2015. HBV has been 45 co-evolving with mammals for millions of years. Recently, the Smc5/6 complex, known for 46 its essential housekeeping functions, was identified as a restriction factor of human HBV 47 antagonized by the regulatory HBx protein. Here, we address whether the antiviral activity of 48 Smc5/6 is an important evolutionary-conserved function. We found that all six subunits of 49 Smc5/6 have been conserved in primates with only Smc6 showing signatures of “evolutionary 50 arms-race”. Using evolutionary-guided functional assays that include infections of primary 51 human hepatocytes, we demonstrate that HBx from very divergent mammalian HBVs could 52 all efficiently antagonize Smc5/6, independently of the host species and sites under positive 53 selection. These findings show that the Smc5/6 antiviral activity against HBV is an important 54 function in mammals. It also raises the intriguing possibility that Smc5/6 restricts other, yet 55 unidentified viruses. 56 3 bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Filleton, Abdul, et al. 57 Introduction 58 Hepatitis B virus (HBV) infects more than 250 million people worldwide and is a 59 leading cause of chronic hepatitis and liver cancer in humans (World Health Organization). 60 HBV is a member of the Hepadnaviridae family of DNA viruses, which have co-evolved 61 with their host species for millions of years (1-3). Today, hepadnaviruses are found to 62 naturally infect, in a species-specific manner, mammals, birds, as well as fish and amphibians. 63 In mammals, HBVs are present in rodents, bats, and several primates including human, 64 chimpanzee, gibbon, orangutan, and the New World wooly monkey. Mammalian 65 hepadnaviruses (orthohepadnaviruses) all contain a gene encoding a small regulatory protein, 66 HBx, that is thought to have arisen de novo in the orthohepadnavirus lineage (1). HBx has 67 long been known to play a central role in HBV replication and pathogenesis (4-6) and has 68 recently been shown to have a key role in promoting HBV transcription by antagonizing the 69 restriction function of the infected cell’s Structural Maintenance of Chromosome (SMC) 70 Smc5/6 complex (7, 8). However, whether this property has been conserved among the HBx- 71 containing hepadnaviruses is unknown. 72 The Smc5/6 complex is, together with cohesin and condensin, one of the three SMC 73 complexes found in eukaryotes (9, 10). As for the other SMC complexes, the core of the 74 Smc5/6 complex is formed by a heterodimer of two SMC proteins, Smc5 and Smc6 (11), 75 which associate with four additional subunits known as non-SMC elements (Nsmce1-4) 76 (Figure 1A). These SMC complexes all have essential housekeeping functions, playing 77 fundamental roles in chromosome replication, segregation and repair (reviewed in (10). 78 Condensin controls chromosome condensation during mitosis and cohesin maintains cohesion 79 between the newly replicated sister chromatids. However, the role of the Smc5/6 complex is 80 less well understood. It has reported functions in DNA replication and repair, but its exact 81 mode of action remains elusive (12-16). 4 bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Filleton, Abdul, et al. 82 In addition to its essential cellular activities, a novel function of human Smc5/6 83 complex as an HBV restriction factor has been recently dissected: in the absence of HBx, the 84 Smc5/6 complex binds to the HBV episomal DNA genome and inhibits viral transcription (7, 85 8, 17). Human HBx antagonizes this effect by high-jacking the host DDB1-containing E3 86 ubiquitin ligase complex to target the Smc5/6 complex for ubiquitin-mediated degradation, 87 thereby enabling productive HBV gene expression (7). 88 Most genes encoding for antiviral restriction factors have been engaged in an 89 “evolutionary arms-race” with the viruses they inhibit (18, 19). Indeed, during long-term co- 90 evolution, pathogenic viruses and their hosts are constantly under the selective pressure of the 91 other for survival. As a result, host restriction factors evolve rapidly and display signatures of 92 positive (diversifying) selection. These signatures can be identified by analyzing the codon 93 sequences of orthologous genes from a large number of related species. At virus-host 94 interaction sites, one can witness adaptive changes, including frequent amino acid changes 95 (where a higher non-synonymous substitution rate, dN, than the synonymous rate, dS, is 96 indicative of positive selection), and insertion/deletions (indels) or splicing variants as ways 97 to modify the virus-host interface and to escape from viral antagonists (19-24). 98 To assess whether the antiviral function of the Smc5/6 complex has been 99 evolutionarily important, we performed phylogenetic and evolutionary analyses of virus and 100 host proteins in combination with functional assays. We found that all six subunits of the 101 Smc5/6 complex have been highly conserved in primate evolution, with only Smc6 showing 102 signatures of an “evolutionary arms-race”. Because orthohepadnaviruses have diverged 103 millions of years ago and their HBx have very little sequence homologies, we then 104 investigated the Smc5/6-antagonism capacity of HBx from six divergent orthohepadnaviruses 105 from primates, rodents and bats. We found that all orthohepadnavirus HBx are efficient at 106 counteracting the Smc5/6 complex, independently of the host species or the variations at sites 5 bioRxiv preprint doi: https://doi.org/10.1101/202671; this version posted December 1, 2017.